Extracellular Volume Fraction Calculated Using Contrast-Enhanced Computed Tomography as a Biomarker of Oxaliplatin-Induced Sinusoidal Obstruction Syndrome: A Preliminary Histopathological Analysis

Background Oxaliplatin (OX)-based chemotherapy induces sinusoidal obstruction syndrome (SOS) in the nontumorous liver parenchyma, which can increase the risk of liver resection due to colorectal liver metastasis (CRLM). The extracellular volume (ECV) calculated from contrast-enhanced computed tomography (CT) has been reported to reflect the morphological change of hepatic fibrosis. The present retrospective study aimed to evaluate the ECV fraction as a predictive factor for OX-induced SOS. Methods Our study included 26 patients who underwent liver resection for CRLM after OX-based chemotherapy with a preoperative dynamic CT of appropriate quality. We investigated the relationship between the pathological SOS grade and the ECV fraction. Results Overall, 26 specimens from the patients were graded with the SOS classification of Rubbia-Brandt et al. as follows: grade 0, n = 17 (65.4%); grade 1, n = 4 (15.4%); and grade 2, n = 5 (19.2%). No specimens showed grade 3 SOS. In a univariate analysis, the ECV fraction in grade 0 SOS was significantly lower than that in grade 1 + 2 SOS (26.3 ± 3.4% vs. 30.6 ± 7.0%; P = 0.025). The cutoff value and AUC value of the ECV fraction to distinguish between grades 0 and 1 + 2 were 27.5% and 0.771, respectively. Conclusions Measurement of the ECV fraction was found to be a potential noninvasive diagnostic method for determining early-stage histopathological sinusoidal injury induced by OX-based chemotherapy.


Introduction
Te liver is the most common site of metastasis of colorectal cancer, and liver metastasis is a major contributor to mortality in patients with this disease. Liver resection currently remains the treatment that ofers the best chance of long-term survival to patients with colorectal liver metastasis (CRLM) and has become the standard of treatment in resectable cases [1,2]. Systemic chemotherapy is the standard treatment for patients with unresectable CRLM.
Perioperative chemotherapy has been proven to efectively downsize metastases, improving disease-free survival in patients with resectable disease [3]. On the other hand, systemic chemotherapy for CRLM has been demonstrated to cause injury to the nontumoral liver parenchyma [4,5]. Oxaliplatin (OX) can induce hepatic sinusoidal obstruction syndrome (SOS), which can lead to thrombocytopenia and severe liver dysfunction [6] and a higher rate of postoperative morbidity [7]. Terefore, it is important to establish an accurate method with which the histopathological change of the liver caused by SOS can be evaluated. Te development of a noninvasive imaging technique would enable wide screening and closer monitoring of patients who are at risk for SOS. So far, few efective imaging fndings have been shown to refect the parenchymal changes in OXinduced SOS. Overman et al. [6] reported that spleen size correlates with increasing grade of hepatic sinusoidal injury. O'Rourke et al. [8] also demonstrated that ferucarbotranenhanced MRI could predict the severity of chemotherapyassociated hepatic cellular injury. As a novel candidate quantitative diagnostic method for SOS, we focused on the measurement of the extracellular volume (ECV) fraction on contrast-enhanced computed tomography (CE-CT), which several studies have reported to have potential application in the diagnosis of liver fbrosis based on clinicopathological analyses [9][10][11] and animal experiments [12].
Te hepatic parenchyma is generally considered to consist of the following three distinct spaces: the intravascular space (IVS), the intracellular space, and the extravascular-extracellular space (EES) [13]. Te histopathological feature of hepatic fbrosis is the expansion of the EES secondary to the deposition of collagen and matrix proteins [14]. While the measurement of the EES is challenging, the quantifcation of the extracellular space (ECS), which is the sum of the EES and the IVS on the CE-CT, is relatively simple. All current water-soluble conventional CT contrast materials pass freely between the EES and the IVS of the liver but are not taken up by living cells [15]. After parenteral injection, the distribution of contrast material reaches an equilibrium in which the concentration of contrast material is similar between the EES and the IVS. Images acquired at this time are commonly referred to as equilibrium-phase images. As the concentration of contrast material on CT is directly proportional to the CT attenuation, the ECV (the volume of the ECS) fraction is simply estimated as the ratio of enhancement of the liver parenchyma to enhancement of the blood pool multiplied by the diference of 1 minus the hematocrit value during the equilibrium phase.
Based on the concept of the ECV fraction, we hypothesized that the ECV fraction increases due to the expansion of the IVS in the liver of the patients with OX-induced SOS, which histopathologically presents with sinusoidal dilatation, whereas the expanding EES leads the ECV fraction to increase with the progression of liver fbrosis [12]. In the present preliminary study, we evaluated the potential of the ECV fraction to estimate the degree of SOS using histopathological analyses of specimens from patients who underwent hepatectomy after chemotherapy with OX.

Study Population.
Between January 2016 and January 2021, 51 consecutive patients underwent surgical liver resection for CRLM in the Department of Surgery, Miyazaki University Hospital, Miyazaki, Japan. A retrospective review of the data for these patients was conducted. Twenty-six (51%) patients who met the inclusion criteria were included in the subsequent analysis. Te inclusion criteria were as follows: systemic chemotherapy containing OX before surgery, adequate preoperative dynamic CT, within 30 days between the preoperative CT and the surgical resection, and sufcient nontumoral liver tissue to perform a representative pathological analysis ( Figure 1).
Tis single-center, retrospective, observational study was performed at Miyazaki University Hospital and approved by the institutional ethical committee (study number: O-1132). Consent was obtained from all participants included in this study via an opt-out format.

CT Protocol.
CT examinations were performed using a 64-row multislice CT system (IQon Spectral CT, Philips Healthcare, Eindhoven, Te Netherlands) and a 320-row multislice CT system (Aquilion ONE Genesis Edition, Tokyo, Japan). After the acquisition of precontrast scans, each subject was injected with a properly selected iodinated contrast agent of one of four brands (Omnipaque, GE Healthcare, Boston, MA; Optiray, Guerbet, Paris, France; Iomeron, Eisai, Tokyo, Japan; or Iopromide, Fujiflm, Tokyo, Japan), using a Dual Shot GX7 power injector (Nemoto, Tokyo, Japan). Te injection dose was 600 mg of iodine per kg of body weight, and the duration was fxed at 30 seconds; hence, the injection rate depended on the patient's body weight. Triple-phasiccontrast-enhanced scans through the abdomen were performed without a bolus tracking program. Equilibrium-phase images were obtained 180 seconds after injection. Te slice thickness for the contrast-enhanced images was 2 mm. Te images were saved in DICOM format and transferred to an image workstation using the SYNAPSE VINCENT software program (Fujiflm).

Image Analysis.
Te image analysis was performed using electronically stored CT images without knowledge of the clinical information. CT images taken within 30 days before surgery were included in this analysis. ECV fraction in all cases was measured by a single clinician with prior guidance and agreement from a radiologist knowledgeable in ECV. Operator-defned regions of interest (ROIs) were used to measure the CT values (in Hounsfeld units, HU) of the liver parenchyma and abdominal aorta on both precontrast and equilibrium-phase images. CT values of the liver parenchyma were quantifed by placing an oval ROI that carefully avoided the tumor, bile duct dilatation, and intrahepatic vessels. Te ROIs were placed on the 3 areas of the lateral segment, S8 and S7. Te size of the ROI was measured in more than 1000 mm 2 of liver parenchyma, with the exclusion of blood vessels. Te hepatic CT-ECV fraction was calculated using the following formula [9][10][11][12]: where ∆HU liver and ∆HU aorta represent absolute enhancement, which were the CT values on the equilibrium-phase image minus the CT values on the precontrast images of the liver parenchyma and abdominal Ao, respectively. All measurements were performed in 3 diferent slices and the mean values were calculated ( Figure 2). Serum hematocrit levels were obtained within 48 h of the CT examination. We calculated the splenic volume (SV) using SYNAPSE VINCENT, as described in a previous report [16]. Te SV was measured before chemotherapy and after chemotherapy before resection of the CRLM. Te rate of increase of the SV was calculated as follows:

Preoperative Assessment of the Liver Function.
Preoperative serum biological data, including the platelet count (PLT, (10 9 /L)), hematocrit value (Ht, (%)), aspartate alkaline phosphatase (AST, (U/L)), alanine aminotransferase (ALT, (U/L)), and type IV collagen, were collected. An indocyanine green (ICG) test was performed as follows: a dose of 50 mg of ICG dissolved in 10 ml of sterile water was injected through a peripheral vein based on the body weight of the patient (0.5 mg/kg). Te retention rate of ICG at 15 minutes (ICG R15, (%)) was calculated with the values expressed as percentages at 5, 10, and 15 minutes after injection. Te AST to platelet ratio index (APRI) score [17] and a score based on the relationships among four regression coefcients (FIB-4 index) [18], which were initially used to predict fbrosis in patients sufering from hepatitis C, were calculated according to the following formulas: APRI score � AST[U/L]/AST upper limit of normal PLT 10 9 /L × 100, 2.5. Histopathological Analysis. All formalin-fxed, parafnembedded samples of nontumoral liver parenchyma were reviewed by one pathologist who was unaware of the clinical and biological patient data. Morphological analyses were conducted using hematoxylin and eosin (H&E)-stained slides. Sinusoidal congestion was graded from 0 to 3 according to the severity of the fndings, as proposed in the original publication by Rubbia-Brandt et al. [4], in which grade 0 � absent, grade 1 � mild (centrilobular involvement limited to one-third of the lobular surface), grade 2 � moderate (centrilobular involvement extending into two-thirds of the lobular surface), and grade 3 � severe (complete lobular involvement) ( Figure 3). Perisinusoidal fbrosis was also analyzed with Masson trichrome-stained slides as previously reported by Rubbia-Brandt et al. [19] and graded from 0 to 2 as follows: grade 0 � absent, grade 1 � mild (<50% sinusoids evaluated on 20 felds at ×200 magnifcation), and grade 2 � moderate (>50% sinusoids evaluated on 20 felds at ×200 magnifcation) ( Figure 4).

Statistical Analysis.
All continuous variables are expressed as the mean ± standard deviation. Te Mann-Whitney U test was used to compare continuous variables. A one-way analysis of variance was used as a multiple comparison test. Categorical variables are summarized as numbers and percentages and were compared between groups using Fisher's exact test or the chi-squared test, as appropriate. A nonparametric receiver operating characteristic curve analysis was used to calculate the area under the receiver operating characteristic curve (AUC). Younden index is used as a criterion for selecting the optimum cutof point. Statistical analyses were performed using StatFlex version 7 (Artech, Osaka, Japan), and P values of <0.05 were considered statistically signifcant.

Correlation of Imaging Parameters with Histopathological
Sinusoidal Dilatation. Te mean ECV fraction of each grade gradually increased from grade 0 to grade 2 sinusoidal dilatation (grade 0: 26.3 ± 3.4%, grade 1: 28.8 ± 2.5%, and grade 2: 32.1 ± 9.4%). However, a multiple comparison test showed   Journal of Oncology no signifcant diference in the correlation of the ECV fraction with the grade of SOS (P � 0.086) ( Figure 5(a)). A multiple comparison test showed a signifcant difference in the correlation of the rate of increase of the SV with the grade of SOS (P � 0.023) ( Figure 5(b)). Te mean rate of increase of the SV in each grade was as follows: 114.2 ± 24.3% in grade 0, 110.3 ± 14.8% in grade 1, and 169.7 ± 70.5% in grade 2.

Discussion
In SOS, histopathological changes are characterized by sinusoidal dilatation with associated hepatocyte atrophy. Te microscopic change is macroscopically identifed as socalled "blue liver" because the congested liver grossly shows a blue color. Rubbia-Brandt et al. [4] frst reported SOS induced by OX-based chemotherapy in the nontumorous specimens of patients undergoing hepatic resection and proposed the hypothesis that an initial toxic injury to the sinusoidal endothelial cells results in sinusoidal wall disruption, which is followed by activation of hepatic stellate cells and the deposition of matrix in the sinusoids. In their retrospective study, they reported that 78% of patients who received OX had some degree of sinusoidal injury and established the invaluable histological grade classifcation mentioned above. A meta-analysis reporting grade 2 or greater sinusoidal injury demonstrated that those receiving OX-based regimens were at a 4.36-fold increased risk of SOS in comparison to chemotherapy-naïve control subjects [20]. According to the analysis, 17.2% of patients with OX-based chemotherapy developed moderate to severe SOS, which was similar to the frequency in our study (19.2%).
Te clinical importance of SOS is refected in the development of hepatomegaly, ascites, splenomegaly, thrombocytopenia, portal hypertension, and systemic elevation of liver enzymes [6,[21][22][23]. In the context of liver surgery, numerous studies have shown a negative infuence of SOS on postoperative outcomes (e.g., postoperative liver failure, higher morbidity rates, and longer hospital stay) [24]. Furthermore, another study demonstrated that, over the long term, SOS could lead to early recurrence and decreased survival [25]. A histological examination of hepatic tissue is the only method that can be used to accurately determine the degree of OX-induced SOS. However, a histological examination is not feasible in the routine diagnosis of patients with suspected SOS, and it remains challenging to reliably determine the presence of SOS (which is essential to continue chemotherapy safely and better select and prepare patients before liver resection) by noninvasive imaging tests.
Several previous studies have identifed predictive factors of OX-induced SOS, including the administration of six or more cycles of chemotherapy [24], the preoperative AST Journal of Oncology 5 value [24], ICG R15 [24], APRI [26], and the FIB-4 index [26]. However, the present study found that the abovedescribed factors do not difer to a statistically signifcant extent between the grades of SOS. Overman et al. [6] hypothesized that changes in spleen size would refect the extent of hepatic sinusoidal injury after OX-based chemotherapy, based on the clinical observation of prolonged thrombocytopenia and splenic enlargement in patients treated with such therapies and the understanding of the mechanism of SOS. In their retrospective study, treatment with adjuvant FOLFOX before hepatectomy resulted in an increase in spleen size in 86% of patients, and an increase in spleen size was strongly correlated with a higher grade of SOS. Te present study found a signifcant infuence of moderate (grade 2) SOS in comparison to no or mild SOS (grades 0-1) on the rate of increase of the SV. On the other hand, no infuence was apparent when mild to moderate SOS (grades 1-2) was compared to no SOS (grade 0). From these results, we considered that splenic enlargement is not a suitable factor for predicting early-stage SOS because it refects the indirect fndings based on the condition in which a more advanced morphological hepatic change causes a sufcient degree of portal hypertension to cause the spleen to enlarge. Tis study is the frst to report that OX-induced SOS can be predicted using the ECV fraction as a noninvasive quantitative parameter calculated based on preoperative CE-CT. Although this parameter was initially described in the setting of hepatitis-related liver fbrosis [27], we herein show its usefulness in the setting of chemotherapy-related liver injury. Another important fnding of our study is that CT-ECV may be able to detect the change of OX-induced SOS at an earlier time point in comparison to the rate of increase of the SV. While this novel imaging parameter has not been observed as a parameter of SOS, there is abundant evidence to support its use as a predictive factor of fbrosis in the myocardium [9], the liver [10,27], and the pancreas [11,28]. Tis study showed an important discovery in that the ECV fraction is a parameter that may be able to predict    Journal of Oncology grade 0 SOS (AUC: 0.771; sensitivity: 0.667; specifcity: 0.706). As a diagnostic parameter for SOS, the ECV fraction is diferent from the rate of increase of the SV in that the ECV fraction is a direct parameter that can be calculated based on the concept that imaging features refect histological changes in the liver, whereas enlargement of the spleen is an indirect parameter that results from the hemodynamic change caused by the change of the hepatic parenchyma. Terefore, more useful diagnostic methods may be established when both of the parameters, which can be calculated from a single routine CE-CT scan, are well combined.
In the patients with OX-induced SOS, the fbroses were known to be proportionally associated with the severity of sinusoidal dilatation [19]. Since the ECV fraction has been correlated with tissue fbrosis in previous papers, we examined tissue fbrosis associated with the progression of SOS. Unlike the previous report, our results revealed that the prevalence of fbrosis was similar in each dilatation grade. Furthermore, no signifcant diference of the ECV fraction was shown between the patients with and without perisinusoidal fbrosis (28.1 ± 6.8% vs. 27.5 ± 2.8%; P � 0.783). Te reason for concern is that the very low prevalence of severe fbrosis (grade 2 fbrosis was only 3.8% overall) had little impact on the ECV fraction. Such characteristics of the patient background might provide certain advantages in evaluating the correlation between ECV and sinusoidal dilatation.
Our study was associated with several important limitations. First, it was a retrospective study of a small number of patients. Second, we used two diferent CT systems. Tird, when extracting CT values, some measurement errors may have occurred due to the manual ROI setting. Based on the results of this preliminary study, it will be necessary to conduct a further prospective study in a larger population using dual-energy CT. Dual-energy CT can accurately extract the iodine concentration in a specifc range without subtracting the CT values of the equilibrium phase from the precontrast phase [29]. Using this imaging device, it should be possible to more accurately determine the CT-ECV fraction.
In conclusion, the ECV fraction was found to have a potential application as a noninvasive diagnostic method for determining histopathological sinusoidal injury induced by OX-based chemotherapy at an early stage. We would like to examine the clinical impact of this novel parameter on surgical outcomes after hepatectomy for CRLM in a highquality prospective study.